Thymidylate synthetase is the target enzyme for the important cancer chemotherapeutic drug 5-fluorouracil. The nature of this enzyme and the interaction of this enzyme with 5-fluorouracil and other drugs has been studied extensively utilizing enzymes isolated from the bacterium Lactobacillus casei. It is the objective of this research to identify a mammalian source of thymidylate synthetase containing sufficient quantities of the enzyme to make purification of relatively large quantities practical. Subsequent to purification, the nature of drug interaction with the mammalian enzyme will be studied. A cultured line of mouse hepatoma cells is currently under investigation as the source of the mammalian enzyme. This cell line is also under investigation for use in establishing a model system to study the introduction of radioactively labelled drug into whole cells and thereby affinity label, in vivo, thymidylate synthetase.